We have previously generated alpha-crystallin gene knockout mice to study the in vivo function of these remarkable proteins. The alpha-crystallins comprise a large fraction of the soluble protein in the vertebrate lens where they were, for many years, believed to function solely as structural proteins. Lenticular alpha-crystallin is comprised of two similar subunits alphaA and alphaB, each encoded by a single gene. They are related to the small heat shock proteins, and in vitro they exhibit molecular chaperone activity, autokinase activity, and interact with, and affect the state of, several cytoskeletal components. alpha-Crystallin, especially alphaB-crystallin, has been shown to be a normal constituent of many non-lenticular tissues, and has been detected in cytoplasmic inclusion bodies found in several human pathological conditions. alphaA-Crystallin, and indeed many of the formerly "lens-specific" crystallins, has also recently been shown to be expressed on non lenticular tissues. Toward understanding the major roles of alpha-crystallin in vivo, we previously generated alphaA- and alphaB-crystallin gene knockout mice and alphaA-/alphaB-crystallin gene double knockout mice (DKO).[unreadable] [unreadable] We have previously shown that the lenses of DKO mice exhibit disintegration of fiber cells surrounding the lens nucleus, and have recently shown that these morphological abnormalities result from elevated DEVDase and VEIDase activities in lenses lacking alpha-crystallin, suggesting involvement of the apoptosis pathway in this pathology. [unreadable] [unreadable] Binding of partially processed or intact procaspase 3 to alphaB-crystallin has already been reported by other laboratories, and our lab recently demonstrated, via pull-down assay, an interaction between caspase 9 and alphaA-crystallin in lens extract and direct interaction between purified caspase 9 and purified alphaA-crystallin. Moreover, alpha-crystallin complex and individual alphaA-crystallin and alphaB-crystallin inhibited, by 10 fold, the caspase 9-mediated cleavage of procaspase 3 to the active form of caspase 3 in a cell-free system, with concomitant formation of 26 kDa polypeptide. This polypeptide is not produced in the canonical maturation of caspase 3, however, accumulation of 24/26 kDa caspase 3 polypeptide, in the presence of alpha-crystallin, has also been reported by other laboratories.[unreadable] [unreadable] Analysis of 6-His N-terminal-tagged procaspase 3 digestion products suggested that, in the presence of alpha-crystallin, caspase 9 acts at an alternative cleavage site, Glu 231. Analysis of the caspase 3 three-dimensional structure reveals that this region is located on the surface of the molecule, and forms a loop between two helices, which is accessible for caspase 9 cleavage. Digestion at this site should result in 26 kDa N-terminal and 5.5 kDa C-terminal peptides. If the precursor or 17 kDa subunit is cleaved at Glu 231, the peptide containing Phe 250, which interacts in the active center, would be separated from active center of caspase 3.[unreadable] [unreadable] The most remarkable results involving alphaB-crystallin this year came from the laboratory of collaborator Dr. Lawrence Steinman. A Nature paper involving a collaboration between the Steinman, van Noort and Wawrousek labs demonstrated that alphaB-crystallin protein, injected intravenously into mice with experimental autoimmune encephalitis, can greatly decrease clinical symptoms in this mouse model of multiple sclerosis (MS). The mechanism by which this protein exerts its curative effect is not yet known. However, this may provide major benefits in future treatments of MS, and the immunomodulatory effects observed may have relevance in other autoimmune-mediated diseases. [unreadable] [unreadable] Dr. Debasish Sinha of Johns Hopkins University is on sabbatical in this laboratory for part of this year and part of the next fiscal year. We are collaborating on an animal modeling project to investigate the non-lens functions of lens betaA3/A1-crystallin. Dr. Sinha and associates have shown that a naturally occurring mutation of this gene causes abnormalities in astrocytes, which leads to altered development of the neural retina and retinal vasculature. We are making mouse models in which expression of the identified mutation is restricted to astrocytes. In collaboration with Dr. Lijin Dong of the NEI Genetic Engineering Facility, we are also generating conditional knockout mice, in which the gene is specifically deleted in astrocytes. Using this approach, we can study the retinal effects of this protein, without interference or complicating secondary effects from a grossly malformed lens. Generation of these mouse models is presently underway.[unreadable] [unreadable] In collaboration with Allen Taylor, we are examining the role of the ubiquitin proteasome pathway in lens development. Transgenic mice created in our lab, expressing a mutant, dominant negative form of ubiquitin in their lenses show a cataract phenotype, and accumulation of ubiquitinated proteins. The cataract varies in severity among various transgenic lines, and to date, the most severe line, in which the lens becomes almost completely destroyed in the adult, has been most thoroughly studied. Although the cataract is always severe in this line, microophthalmia is observed only in a portion of the eyes. Ontogeny of the cataract, and impeded development of the lens during early development are currently being studied. [unreadable] [unreadable] In a collaborative effort between this lab and the lab of Melinda Duncan, we demonstrated that a mutation in the gene encoding lens beaded filament Cp49 is present in FVB/N mice, a popular mouse for transgenic work. This mutation results in an almost complete loss of lens beaded filaments, a lens-specific form of cytoskeletal intermediate filaments. This mutation, originally discovered in 129S strains of mice, the strain most used in gene targeting strategies, could potentially complicate interpretation of eye pathology in mouse models generated in this strain. Our finding that the same mutation is present in the FVB/N strain, one of the most popular strains for generating transgenic mice, extends the caution to many transgenic mouse lines that have been produced over the last 25 years in this strain of mouse.[unreadable] [unreadable] A collaboration with David Hinton, Ram Kannan and colleagues is exploring the role of alpha-crystallin in the retina and RPE. Results published this year demonstrate that absence of either alphaA- or alphaB-crystallin increases the susceptibility of RPE cells to peroxide-induced apoptosis, with concomitant induction of caspase 3 activity. Moreover, in peroxide-treated normal RPE, alphaB levels decreased in both cytoplasm and mitochondria in a dose dependent manner, and the mitochondrial levels of both alphaA and alphaB decreased under increasing oxidative stress. The presence of mitochondria-associated alpha-crystallin appears to play a major role in protection of the RPE from oxidative stress, a condition common in this area of the eye.